Antibodies to human b7x for treatment of metastatic cancer

ABSTRACT

Methods are provided for treating metastatic cancer in patients having metastatic cancer or for preventing metastasis in cancer patients at risk for metastasis comprising administering to the patient an antibody to B7x, or an active antibody fragment that binds B7x, in an amount effective to treat or prevent metastasis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/252,267, filed Aug. 31, 2016, which is a divisional of U.S. patentapplication Ser. No. 14/050,512, filed Oct. 10, 2013, now U.S. Pat. No.9,447,186, which is continuation-in-part of and claims priority of PCTInternational Patent Application No. PCT/US2012/034348, filed Apr. 20,2012, which designates the United States of America and which claims thebenefit of U.S. Provisional Patent Application No. 61/477,729, filedApr. 21, 2011, the contents of which are herein incorporated byreference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberDK083076 awarded by the National Institutes of Health and grant numberW81XWH-10-1-0318 awarded by the United States Army Medical Research andMateriel Command. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to insuperscripts. Full citations for these references may be found at theend of the specification. The disclosures of these publications arehereby incorporated by reference in their entirety into the subjectapplication to more fully describe the art to which the subjectinvention pertains.

Cancer is a serious public health problem in the U.S. and othercountries. More than 90% of cancer patient deaths result from cancermetastasis rather than from a primary cancer. There are about 924,310new cancer cases and 339,150 cancer deaths in the U.S. alone. Accordingto Cancer Statistics 2010, in the U.S. in 2010 alone, there are anestimated 222,520 new cases and 157,300 deaths for lung cancer, 217,730new cases and 32,050 deaths for prostate cancer, 207,090 new cases and39,840 deaths for breast cancer, 145,500 new cases and 51,370 deaths forgut cancer, 58,240 new cases and 8,210 deaths for kidney cancer, and51,350 new cases and 36,800 deaths for pancreas cancer. Whiletraditional therapies such as surgery, chemotherapy, and radiation canoften control primary cancer growth, successful control of disseminatedmetastases of cancer remains rare.

Cancer and the immune system have dynamic interactions, which playcrucial roles in determining tumor development and thus clinicaloutcome. T cells of the immune system are the major combatants againstcancers. T cell activation, proliferation, differentiation to effectorfunction and memory generation are determined by both positivecostimulation and negative coinhibition, generated mainly by theinteraction between the B7 family and their receptor CD28 family (FIG.1). In 2003, B7x was discovered as a new member of the B7/CD28 family¹.B7x inhibits T cell function in vitro¹. B7x is extremely conservativewith 87% amino acid identity between human and mice.

A study of 823 prostatectomy patients for whom a minimum of 7 yearfollow-up data were available revealed that prostate cancer patientswith strong expression of B7x by tumor cells were significantly morelikely to have disease spread at time of surgery, and were atsignificantly increased risk of clinical cancer recurrence andcancer-specific death². In addition, all of 103 ovarian borderlinetumors tested expressed B7x³. In contrast, only scattered B7x-positivecells were detected in non-neoplastic ovarian tissues. Otherinvestigators have reported that B7x is over-expressed in human cancersof the lung⁴, ovary⁵, breast^(6,7), kidney⁸, brain⁹, pancreas¹⁰,esophagus¹⁷, skin¹⁸, gut³⁶, stomach¹⁹ and thyroid³⁵. In renal cellcarcinoma, patients with tumors expressing B7x were three times morelikely to die from renal cancer compared to patients lacking B7x⁸. Inhuman breast cancer, there was a significant association between a highproportion of B7x positive cells in invasive ductal carcinomas anddecreased number of tumor infiltrating lymphocytes. In esophagealsquamous cell carcinoma, expression levels of B7x on tumor cells weresignificantly correlated with distant metastasis, tumor stage and poorsurvival, and were inversely correlated with densities of CD3+ T cellsin tumor nest and CD8+ T cells in tumor stroma.

The present invention addresses the serious and long-felt need fortreatments for metastatic cancer.

SUMMARY OF THE INVENTION

Methods are provided for treating metastatic cancer in a patient havingmetastatic cancer or for preventing metastasis in a cancer patient atrisk for metastasis comprising administering to the patient an antibodyto B7x, or an active antibody fragment that binds B7x, in an amounteffective to treat or prevent metastasis.

Methods are also provided for treating metastatic cancer in a patienthaving metastatic cancer or for preventing metastasis in a cancerpatient at risk for metastasis comprising determining the level of B7xexpression in a tumor sample from the patient, and if B7x isover-expressed in the tumor sample compared to healthy tissue,administering to the patient an antibody to B7x, or an active antibodyfragment that binds B7x, in an amount effective to treat or preventmetastasis.

Methods are also provided for producing a monoclonal antibody to B7xcomprising immunizing a B7x knockout mouse with a B7x-Ig fusion protein,generating a hybridoma from spleen cells from the mouse, and testingsupernatant from the hybridoma for its ability to react with immobilizedB7x-Ig or a cell line expressing B7x, but not with control Igs or celllines expressing other B7 family members, to identify a monoclonalantibody to B7x.

Methods are further provided for screening monoclonal antibodies to B7xto identify an antibody that inhibits tumor growth in vivo comprisinginjecting tumor cells expressing B7x on their cell surface into a mouseto induce a tumor in the mouse, and injecting a monclonal antibody toB7x into the mouse to identify an antibody that inhibits tumor growth invivo.

Methods are in addition provided for preventing reoccurrence of a tumorin a patient comprising administering to the patient an antibody to B7x,or an active antibody fragment that binds B7x, in an amount effective toprevent reoccurrence of a tumor in a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The B7 family/CD28 family. The members of the B7 and CD28families have been divided into three groups by phylogenetic analysis:group I includes the pathway of B7-1/B7-2/CD28/CTLA-4, and the pathwayof B7h/ICOS; group II consists of the pathway of PD-L1/PD-L2/PD-1; andgroup III contains B7-H3 and B7x, whose receptors are currently unknown.Modified from Zang et al.¹

FIG. 2A-2B. Expression of B7x on tumor CT26 (A) or MC38 (B) acceleratesdisease progression in vivo. FACS analysis with PE-anti-B7x (line) orcontrol PE-Ab (solid) showed that tumor cell lines CT26 and MC38 did notexpress B7x and that transfectants B7x/CT26 and B7x/MC38 expressedabundant cell surface B7x. Survival curves showed that syngeneic mice ivinjected with B7x/CT26 or B7x/MC38 died much faster than mice injectedwith CT26 or MC38. The log-rank test was used for statistical analyses.P-values<0.05 were considered statistically significant.

FIG. 3. Syngeneic BLAB/c mice iv injected with CT26 or B7x/CT26 andkilled on day 20 for determining tumor nodules in lungs. Mice receivingB7x/CT26 had more tumor nodules than mice receiving CT26, P=0.0002.

FIG. 4A-4C. B7x deficiency protects mice from lung metastasis of cancer.After intravenous injection with breast cancer cell line 4T1, B7xknock-out (B7x−/−) mice had fewer tumor nodules than Balb/c wildtype(B7x+/+) mice at day 18 (A); all wildtype mice were dead within 36 dayswhereas 33% of B7x−/− mice were still alive at day 72 (B). SurvivingB7x−/− mice were still alive at day 140 after rechallenge with 4T1(B),and no tumor was found in lungs from surviving B7x−/− mice with 4T1double-challenge. Wildtype mice had more regulatory T cells andmyeloid-derived suppressor cells in lungs than B7x−/− mice during lungmetastasis of cancer (C).

FIG. 5. Monoclonal antibody clones 12D11 and 1H3 reduced more than 50%tumor nodules in a lung metastasis of cancer model.

FIG. 6. BLAB/c mice iv injected with CT26 or hB7x/CT26 and killed on day17 for determining tumor nodules in lungs. Mice receiving hB7x/CT26 hadmore tumor nodules than mice receiving CT26, P=0.02.

FIG. 7. Anti-B7x monoclonal antibody 1H3 reduced more than 60% of tumornodules in a human B7x-expressed lung metastasis of cancer model.

FIG. 8. Antibody-dependent cell-mediated cytotoxicity (ADCC) assayshowed that three mAbs (1H3, 12D11, 15D2), but not 37G9, were able tokill tumor cells by ADCC mechanism.

FIG. 9. Possible mechanisms by which treatment with anti-B7x antibodiesinhibit metastatic cancer progression in vivo (see Experimental Detailsfor discussion).

FIG. 10A-10D. The effect of anti-B7x monoclonal antibodies on tumorgrowth and survival of mice. (A) BALB/c mice were iv injected with CT26cells expressing mouse B7x (B7x/CT26) at day 0 and then with anti-B7xmAbs 12D11 and 1H3 or control mouse IgG. After sacrifice, tumor nodulesin lungs were counted. Data were pooled from three independentexperiments (n=9 or 10). (B) BALB/c females were injected with 4T1 cellsexpressing mouse B7x (B7x/4T1) in the mammary fatpad. Mice were iptreated with mAb 1H3. After mice were sacrificed, breast tumors inducedtumor nodules on lungs were counted (n=10). (C) BALB/c mice were ivinjected with CT26 cells expressing human B7x (hB7x/CT26) at day 0 andthen injected ip with mAb 1H3 or control mouse IgG. After micesacrifice, tumor nodules in lungs were counted (n=9). Results werepooled from two independent experiments. (D) BALB/c mice were ivinjected with B7x/CT26 at day 0 and then injected ip with 1H3 or controlmouse IgG. At day 60 post-injection, remaining mice were ivre-challenged with B7x/CT26 (n=10).

FIG. 11A-11G. Anti-B7x therapy alters the intratumor balance ofanti-tumor effector immune cells and immunosuppressive cells. BALB/cmice were iv injected with B7x/CT26 and then treated with 1H3 or controlmouse IgG. At day 17, single-cell suspensions from tumor bearing lungswere FACS analyzed for percentage of infiltrated CD45+ cells (A), CD8 Tcells and NK cells (B), tumor antigen AH1 (SPSYVYHQF (SEQ IDNO:5))-specific CD8 T cells (C), CD4 T cells that were Tim-3+PD-1+,Tim-3+ alone and PD-1+ alone (D), and CD11b+Ly6C+ monocyticmyeloid-derived suppressor cells (F). Cell suspensions from tumorbearing lungs were stimulated with 1× cell stimulation cocktail for 5hours and stained with antibodies to CD3, CD4 and IFN-γ or isotypecontrols (E). Shown are the ratios of Treg (CD4+Foxp3+) and MDSCs to CD8T cells, CD4 T cells, and NK cells (G). Results are pooled from threeindependent experiments; *p<0.05, **p<0.01, ***p<0.001.

FIG. 12A-12C. The effects of 1H3 on tumor microenvironments. Paraffinsections of tumor bearing lungs from IgG- and 1H3-treated mice werestained with anti-VEGF and anti-CD31 antibodies. Hematoxylin was usedfor counter-staining. Total amount of VEGF and TGF-β from tumor bearinglungs were measured using ELISA. Each group contained 5 mice. *p<0.05;**p<0.01.

FIG. 13A-13C. Anti-tumor mechanisms of 1H3. (A) 1H3 kills tumor cellsthrough antibody-dependent cellular cytotoxicity. B7x/CT26 cells labeledwith CFSE and PKH-26 were incubated with splenocytes in the presence ofvarious concentrations of mAb 1H3 or IgG. Percentages of tumor celldeath induced by 1H3-mediated ADCC were normalized to those of controlmouse IgG. Data are representative of two independent experiments intriplicates and shown as mean±SE. (B) BALB/c mice were iv injected withB7x/CT26 and then treated with 1H3 or control mouse IgG. After micesacrifice at day 17, lung sections were subjected to the TUNEL assay.Normalized apoptotic staining was measured and compared. *p<0.05. (C)Comparison of therapeutic efficacies between 1H3 and its Fab. BALB/cmice were iv injected with B7x/CT26 cells at day 0 and then injected ip200 μg/mouse with 1H3, Fab of 1H3, or control mouse IgG at day 1, 3, 7,11 and 14. At day 17, tumor nodules in lungs were counted. Data werepooled from two independent experiments (n=9 or 10). *p<0.05;****p<0.0001.

FIG. 14A-14B. Anti-B7x antibody therapy is better than anti-PD-1antibody therapy in two lung metastasis of cancer models. (A) BALB/cmice were iv injected with B7x/CT26 tumor on day 0 and then ip injectedwith normal IgG (control), anti-B7x mAb 1H3, or anti-PD-1 mAb RMP1-14 onday 1,3,7,11, 14. On day 17, mice were sacrificed and numbers of lungtumor nodules were evaluated. Anti-B7x treatment reduced more than 58%of lung tumor nodules, ***P<0.001; whereas anti-PD-1 treatment reducedonly 34% of lung tumor nodules and did not reach statisticalsignificance. (B) BALB/c female mice were injected with B7x/4T1 tumorinto the mammary fatpad on day 0 and then ip injected with normal IgG(control), anti-B7x mAb 1H3, or anti-PD-1 mAb RMP1-14 on day 8,11,13,15,18. On day 20, mice were sacrificed and numbers of lung tumor noduleswere evaluated. Anti-B7x treatment reduced more than 58% of lung tumornodules, *P<0.05; whereas anti-PD-1 did not have an effect.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating metastatic cancerin a patient having metastatic cancer or for preventing metastasis in acancer patient at risk for metastasis comprising administering to thepatient an antibody to B7x, or an active antibody fragment that bindsB7x, in an amount effective to treat or prevent metastasis.

As used herein “metastasize” means, in regard to a cancer or tumor, tospread from one organ or tissue of a patient to another non-adjacentorgan or tissue of the patient.

To “treat” a metastatic cancer means to reduce the number of metastasesin an organ or tissue, and/or to kill metastatic tumor cells or tumorcells that are likely to metastasize, and/or to prevent or reduce thespread of cancerous cells from an original site in the body to anothersite in the body, and/or to inhibit the progression of metastaticcancer, and/or to prevent the reoccurrence of metastasis. Preferably,administration of the antibody or antibody fragment decreases the numberof tumor nodules in the patient. Preferably, administration of theantibody or antibody fragment increases the patient's survival time.

Various diagnostic procedures have been developed to identify cancerpatients at risk for metastasis. See, for example, U.S. PatentApplication Publication Nos. 2008/0138805, 2011/0059470 and2011/0059470, the contents of which are herein incorporated byreference.

The cancer can be, for example, a cancer of the skin, breast, pancreas,prostate, ovary, kidney, espophagus, gastrointestional tract, colon,brain, liver, lung, head and/or neck. In a preferred embodiment, thecancer is lung cancer.

Preferably, the tumor in the patient expresses B7x or overexpresses B7xcompared to healthy tissue, for example, in comparison to healthy tissueof the breast, pancreas, prostate, ovary, kidney, gastrointestionaltract, colon, brain, liver, lung, head and/or neck. The level of B7xexpression in the tumor can be determined, for example, usingimmunohistocytochemistry on a tissue sample obtained from the tumor(e.g., Zang et al.^(2,3)). B7x expression can also be determined bymeasuring expression of B7x mRNA in the tumor sample, for example byNorthern blot hybridization (e.g., Allison et al.¹⁶).

Accordingly, the invention also provides a method for treatingmetastatic cancer in a patient having metastatic cancer or forpreventing metastasis in a cancer patient at risk for metastasiscomprising determining the level of B7x expression in a tumor samplefrom the patient, and if B7x is over-expressed in the tumor samplecompared to the level of B7x expression in healthy tissue, administeringto the patient an antibody to B7x, or an active antibody fragment thatbinds B7x, in an amount effective to treat or prevent metastasis. Themethod can further comprise obtaining a tumor sample from the patient.

As used herein, the term “antibody” refers to complete, intactantibodies. A “fragment” of an antibody refers to a fragment that bindthe antigen of interest, B7x. Antibody fragments include, but are notlimited to, F(ab′)₂ and Fab′ fragments and single chain antibodies.F(ab′)₂ is an antigen binding fragment of an antibody molecule withdeleted crystallizable fragment (Fc) region and preserved bindingregion. Fab′ is ½ of the F(ab′)₂ molecule possessing only ½ of thebinding region. Complete, intact antibodies include, but are not limitedto, polyclonal antibodies, monoclonal antibodies such as murinemonoclonal antibodies, chimeric antibodies, human antibodies, andhumanized antibodies.

Various forms of antibodies may be produced using standard recombinantDNA techniques. For example, “chimeric” antibodies may be constructed,in which the antigen binding domain from an animal antibody is linked toa human constant domain (an antibody derived initially from a nonhumanmammal in which recombinant DNA technology has been used to replace allor part of the hinge and constant regions of the heavy chain and/or theconstant region of the light chain, with corresponding regions from ahuman immunoglobulin light chain or heavy chain). Chimeric antibodiesreduce the immunogenic responses elicited by animal antibodies when usedin human clinical treatments. In addition, recombinant “humanized”antibodies may be synthesized. Humanized antibodies are antibodiesinitially derived from a nonhuman mammal in which recombinant DNAtechnology has been used to substitute some or all of the amino acidsnot required for antigen binding with amino acids from correspondingregions of a human immunoglobulin light or heavy chain. That is, theyare chimeras comprising mostly human immunoglobulin sequences into whichthe regions responsible for specific antigen-binding have been inserted.For example, human IgG Fc can be used to replace the mouse Fc part of amonoclonal antibody.

The antibody can be, e.g., any of an IgA, IgD, IgE, IgG, or IgMantibody. The IgA antibody can be, e.g., an IgA1 or an IgA2 antibody.The IgG antibody can be, e.g., an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4antibody. A combination of any of these antibodies subtypes can also beused. One consideration in selecting the type of antibody to be used isthe desired serum half-life of the antibody. IgG has a serum half-lifeof 23 days, IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days¹⁵.Another consideration is the size of the antibody. For example, the sizeof IgG is smaller than that of IgM allowing for greater penetration ofIgG into tumors. Preferred antibodies include IgG1 monclonal antibodies.

The antibodies and antibody fragments of the present invention aredesigned by humans and made outside of the human body. The antibodiesand antibody fragments are specific for B7x, but not for other B7 familymembers (i.e., B7-1, B7-2, B7h, PD-L1, PD-L2, and B7-H3).

Preferred antibodies include monoclonal antibodies 1H3 and 12D11.Preferred antibodies include monoclonal antibodies having a light and/orheavy chain the same as the light and/or heavy chain of 1H3 or 12D11.The heavy and/or light chains can contain conservative amino acidsequence modifications that do not significantly affect the bindingcharacteristics of the antibody. Preferred antibodies include monoclonalantibodies that bind to the same epitope on B7x as 1H3 or 12D11.Preferred antibodies include monoclonal antibodies that block theinteraction between B7x and its receptor. Preferred antibodies includeantibodies that kill tumor cells through antibody-dependentcell-mediated cytotoxicity. Preferably, the antibody or antibodyfragment blocks inhibition of T cell function by B7x. Preferably, theantibody or antibody fragment blocks interaction betweencancer-expressed B7x and activated T cells. Preferably, the antibody orantibody fragment binds cancer-expressed B7x and kill cancer cells.

The antibodies and antibody fragments used for therapy in the presentinvention do not include antibody-partner molecule conjugates, where thepartner can be, for example, one or more of a drug, toxin, radioisotope,therapeutic agent, or marker agent. The antibodies and antibodyfragments of the present invention are therapeutically effective withoutthe need for another therapeutic agent conjugated to the antibody orfragment. Antibodies and antibody fragments that are used fordetermining the expression of B7x expression in a tumor sample from apatient can be conjugated to a marker, such as, for example, afluorescent marker or a radioisotope marker.

In one embodiment, the antibody or antibody fragment is the soletherapeutic anti-cancer agent administered to the patient. In anotherembodiment, the antibody or antibody fragment can be administered incombination with another anti-cancer agent that is not bound to theantibody or antibody fragment. Anti-cancer agents include, but are notlimited to, an antibody against CTLA-4 (YERVOY®); an antibody againstPD-1 (MDX-1106); an anti-epidermal growth factor receptor (EGFR) agentsuch as, e.g., panitumumab, the anti-EGFR antibody cetuximab (Erbitux®),and the EGFR tyrosine kinase (TK) inhibitors gefitinib (Iressa®) anderlotinib (Tarceva®); an alkylating agent such as, e.g., cisplatin,carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatintetranitrate, mechlorethamine, cyclophosphamide, chlorambucil andifosfamide; paclitaxel and docetaxel; and topoisomerase inhibitors suchas, e.g., irinotecan, topotecan, amsacrine, etoposide, etoposidephosphate and teniposide.

The antibody or antibody fragment can be screened for efficacy using,e.g., procedures set forth herein in Experimental Details.

The antibody or antibody fragment can be administered to the subject ina pharmaceutical composition comprising a pharmaceutically acceptablecarrier. Examples of acceptable pharmaceutical carriers include, but arenot limited to, additive solution-3 (AS-3), saline, phosphate bufferedsaline, Ringer's solution, lactated Ringer's solution, Locke-Ringer'ssolution, Krebs Ringer's solution, Hartmann's balanced saline solution,and heparinized sodium citrate acid dextrose solution. Thepharmaceutical composition can be formulated for administration by anymethod known in the art, including but not limited to, directadministration to a tumor, parenteral administration, intravenousadministration, and intramuscular administration.

The patient can be a human or another animal.

Human and mouse B7x have the amino acid and nucleic sequences indicatedbelow.

Human B7x amino acid sequence (SEQ ID NO: 1):MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMERGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK Mouse B7x amino acid sequence (SEQ ID NO: 2):MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLSCCLMLR. Nucleic acid encoding Human B7x (SEQ ID NO: 3):atggcttccc tggggcagat cctcttctgg agcataatta gcatcatcat tattctggct 60ggagcaattg cactcatcat tggctttggt atttcaggga gacactccat cacagtcact 120actgtcgcct cagctgggaa cattggggag gatggaatcc tgagctgcac ttttgaacct 180gacatcaaac tttctgatat cgtgatacaa tggctgaagg aaggtgtttt aggcttggtc 240catgagttca aagaaggcaa agatgagctg tcggagcagg atgaaatgtt cagaggccgg 300acagcagtgt ttgctgatca agtgatagtt ggcaatgcct ctttgcggct gaaaaacgtg 360caactcacag atgctggcac ctacaaatgt tatatcatca cttctaaagg caaggggaat 420gctaaccttg agtataaaac tggagccttc agcatgccgg aagtgaatgt ggactataat 480gccagctcag agaccttgcg gtgtgaggct ccccgatggt tcccccagcc cacagtggtc 540tgggcatccc aagttgacca gggagccaac ttctcggaag tctccaatac cagctttgag 600ctgaactctg agaatgtgac catgaaggtt gtgtctgtgc tctacaatgt tacgatcaac 660aacacatact cctgtatgat tgaaaatgac attgccaaag caacagggga tatcaaagtg 720acagaatcgg agatcaaaag gcggagtcac ctacagctgc taaactcaaa ggcttctctg 780tgtgtctctt ctttctttgc catcagctgg gcacttctgc ctctcagccc ttacctgatg 840ctaaaataa 849 Nucleic acid encoding mouse B7x (SEQ ID NO: 4):atggcttcct tggggcagat catcttttgg agtattatta acatcatcat catcctggct 60ggggccatcg cactcatcat tggctttggc atttcaggca agcacttcat cacggtcacg 120accttcacct cagctggaaa cattggagag gacgggaccc tgagctgcac ttttgaacct 180gacatcaaac tcaacggcat cgtcatccag tggctgaaag aaggcatcaa aggtttggtc 240cacgagttca aagaaggcaa agacgacctc tcacagcagc atgagatgtt cagaggccgc 300acagcagtgt ttgctgatca ggtggtagtt ggcaatgctt ccctgagact gaaaaacgtg 360cagctcacgg atgctggcac ctacacatgt tacatccgca cctcaaaagg caaagggaat 420gcaaacctag agtataagac cggagccttc agtatgccag agataaatgt ggactataat 480gccagttcag agagtttacg ctgcgaggct cctcggtggt tcccccagcc cacagtggcc 540tgggcatctc aagtcgacca aggagccaac ttctcagaag tctcgaacac cagctttgag 600ttgaactctg agaatgtgac catgaaggtc gtatctgtgc tctacaatgt cacaatcaac 660aacacatact cctgtatgat tgaaaatgac attgccaaag ccactgggga catcaaagtg 720acagattcag aggtcaaaag gcggagtcag ctgcagctgc tcaactccgg gccttccccg 780tgtgtttttt cttctgcctt tgcggctggc tgggcgctcc tatctctctc ctgttgcctg 840atgctaagat ga 852

The invention further provides an antibody to B7x, or an active antibodyfragment that binds B7x, for use in a method of treatment of metastaticcancer in a patient having metastatic cancer or for use in a method ofprevention of metastasis in a cancer patient at risk for metastasis. Theinvention still further provides an antibody to B7x, or an activeantibody fragment that binds B7x, for use in a method of determining ifB7x is over-expressed in a tumor sample from a patient compared tohealthy tissue, and for use in a method of treatment of metastaticcancer in a patient having metastatic cancer or for use in a method ofprevention of metastasis in a cancer patient at risk for metastasis,where B7x is over-expressed in a tumor sample from the patient.

Preferably, the antibody or antibody fragment binds to the IgV domain ofB7x and/or to amino acid residues 35-148 of B7x (e.g., to amino acids35-148 of SEQ ID NO:1).

Preferably, administration of the antibody or antibody fragment preventsthe reoccurrence of a tumor in the patient.

The invention also provides a method of producing a monoclonal antibodyto B7x comprising immunizing a B7x knockout mouse with a B7x-Ig fusionprotein, generating a hybridoma from spleen cells from the mouse, andtesting supernatant from the hybridoma for its ability to react withimmobilized B7x-Ig or a cell line expressing B7x, but not with controlIgs or cell lines expressing other B7 family members, to identify amonoclonal antibody to B7x. Control Igs include, for example, otherB7-Igs such as B7-1-Ig, B7-2-Ig, B7h-Ig, PD-L1-Ig, PD-L2-Ig andB7-H3-Ig. Cell lines expressing other B7 family members include, forexample, cell lines expressing B7-1, B7-2, B7h, PD-L1, PD-L2 and/orB7-H3. Supernatant from the hybridoma can be tested using, for example,an enzyme-linked immunosorbent assay (ELISA) or a fluorescence-activatedcell sorter (FACS). The method can further comprise purifying theantibody from the supernatant. The method can further comprisegenerating a B7x knockout mouse. A B7x knockout mouse can be generated,for example, as follows. 3.4- and 5.2-kb of B7x genomic fragments can becloned into a knock-out vector as 5′ and 3′ arms. The vector can then beelectroporated into embryonic stem cells. An embryonic stem cell cloneheterozygous for the mutant can then be microinjected into blastocystsfrom normal mice. Chimeric males can be crossed with females to giverise to the mutant B7x allele.

The invention further provides a method of screening monoclonalantibodies to B7x to identify an antibody that inhibits tumor growth invivo, the method comprising injecting tumor cells expressing B7x ontheir cell surface into a mouse to induce a tumor in the mouse, andinjecting a monclonal antibody to B7x into the mouse to identify anantibody that inhibits tumor growth in vivo. B7x can be stably expressedon tumor cells using, for example, a retroviral expression vector. B7xexpression on the surface of tumor cells can be confirmed, for example,with antibody to B7x using a fluorescence-activated cell sorter (FACS).Preferably, the antibody inhibits metastasis.

The invention further provides a method for preventing reoccurrence of atumor in a patient comprising administering to the patient an antibodyto B7x, or an active antibody fragment that binds B7x, in an amounteffective to prevent reoccurrence of a tumor in a patient. Therapy withthe antibody or antibody fragment may, for example, increase localinfiltration of anti-tumor immune cells such as CD8 T cells includingtumor antigen-specific CD8 T cells, NK cells, and IFN-γ-producing CD4 Tcells. In addition, or instead, the therapy may, for example, reducelocal infiltration of immunosuppressive myeloid-derived suppressor cells(MDSCs).

This invention will be better understood from the Experimental Details,which follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS Example I Cancer-Expressed B7x Promotes MetastaticCancer Progression

The over-expression of B7x by tumor cells raises the possibility that itmight provide a mechanism by which tumor cells avoid destructionmediated by tumor-reactive T cells and other immune cells. This is aninteresting possibility for two reasons. The first is that high levelsof B7x expression might facilitate tumor progression, and it maytherefore provide a useful prognostic marker to predict outcome. Thesecond is that B7x may be useful therapeutic target for checkpointblockade with immunotherapy.

In order to develop new drugs with B7x as a target, a mouse system wasfirst developed in which cancer-expressed B7x can promote cancerprogression and drugs can be screened. Two lung metastatic cancer modelswere developed in which the expression of B7x on CT26 and MC38 cancercells significantly increased mortality in mice (FIG. 2).

Like most tumor cell lines which permanently lose endogenous B7x proteinexpression after in vitro culture, two tumor lines CT26 and MC38 are B7xnegative and are able to induce lung metastasis when direct injectedintravenously (iv) through the tail vein. A retroviral expression vectorB7x/MSCV was generated to make CT26 and MC38 stably expressing B7x.Syngeneic Balb/c mice were then iv injected with 1×10⁵/per-mouse of CT26or B7x/CT26, and survival kinetics were monitored. Mice injected withCT26 started to die at day 26 and half were still alive at day 50,whereas mice injected with B7x/CT26 started to die at day 21 and wereall dead at day 33 (FIG. 2A). In a parallel study, syngeneic C57BL/6started to die at day 44 after iv injection with 1×10⁵/per-mouse of MC38and 37% were still alive at day 100, whereas mice iv injected with1×10⁵/per-mouse of B7x/MC38 started to die at day 33 and were all deadat day 80 (FIG. 2B).

In a subsequent experiment, Balb/c mice were iv injected with1×10⁵/per-mouse of CT26 or B7x/CT26 and killed on day 20 to determinetumor nodules in the lung. FIG. 3 showed that B7x/CT26 resulted in muchmore tumor nodules in the lung than CT26. Collectively, these resultsdemonstrate that expression of B7x on tumor cells accelerates diseaseprogression in vivo.

Host Cell-Expressed B7x Promotes Expansion of Immune Suppressor Cellswithin the Tumor Microenvironment

Some tissue cells express very low level of B7x, so B7x gene knock-outmice were used to dissect the role of host B7x in cancer immuneresponses. Naïve B7x−/− mice were normal and healthy. B7x-negative 4T1cells (1×10⁵/per mouse) were injected iv into both B7x−/− (on pureBALB/c background) and wildtype BALB/c mice, and tumor progression andtumor nodules in lungs were monitored. 4T1 was B7x negative in vitro andin vivo by RT-PCR (data not shown). At day 18, B7x−/− mice had manyfewer tumor nodules in lungs than wildtype control (FIG. 4A). Allwildtype mice were dead at day 36, whereas 33% of B7x−/− mice were stillalive at day 72 (FIG. 4B). These results reveal that host B7x promoteslung metastasis of cancer and that removal of host B7x protects micefrom this disease. As more than 30% of B7−/− mice survived lungmetastasis, these surviving B7x−/− mice were re-challenged with a higherdose (2×10⁵/per mouse) of 4T1, and their survival was monitored.Remarkably, none of these mice died (FIG. 4B). At day 140 after primaryinjection, these mice were sacrificed and histological examinationdemonstrated that lungs of these mice did not have tumor. These resultssuggest that B7x deficiency not only protects mice from lung metastasisbut also helps to develop strong immune memory of sufficient magnitudeto completely eliminate the tumor.

Flow cytometric analysis was used to identify host cells of tumorinfiltrates that are involved in anti-tumor responses. Wildtype mice hadsignificantly more Foxp3+CD4+ regulatory T cells (Treg) and CD11b+Ly6G+myeloid-derived suppressor cells (MDSC) in the lung than B7x−/− mice onday 18 after iv injection of 4T1 tumor cells (FIG. 4C). Recent work hasrevealed that Treg^(11,12) and MDSC^(13,14) are two major cellpopulations capable of suppressing immune responses. The present resultssuggest that host cell-expressed B7x promotes expansion of immunesuppressor cells within the tumor microenvironment.

Generation of Monoclonal Antibodies Recognizing Both Human and Mice B7x

Monoclonal antibodies (mAbs) were generated against both human and miceB7x from B7x gene knock-out mice. Briefly, B7x knock-out mice wereimmunized with B7x-Ig fusion protein and their spleen cells were usedfor generation of hybridomas. Thirty-two independent clones wereobtained whose supernatant reacted with immobilized B7x-Ig, but notcontrol Ig, in a standard ELISA. Eight of these were selected forfurther study. The specificity of these hybridomas for B7x was confirmedby flow cytometry staining cell lines overexpressing B7x, but not cellsexpressing other B7 family members (B7-1, B7-2, B7h, PD-L1, PD-L2, andB7-H3).

Monoclonal Antibodies Inhibit Metastatic Cancer Progression In Vivo

Four mAbs [clones: 37G9 (IgG2b), 1H3 (IgG1), 12D11(IgG1), 19D6 (IgG1)]were purified and their therapeutic effects examined with B7x/CT26 lungmetastatic cancer model. Syngeneic BLAB/c mice iv injected with1×10⁵/per-mouse of B7x/CT26 on day 0 and then i.p injected with200m/per-mouse of each mAb on day 1,3,7,11,14, or PBS as the control.All mice were killed on day 17 and tumor nodules in each lung wereexamined. Two clones, 12D11 and 1H3, had very good therapeutic effects.Both mAbs were able to reduce more than 50% tumor nodules in the lung insuch a robust metastatic cancer model (FIG. 5). The other two mAb clones(37G9 and 19D6) did not have significant therapeutic effects in vivo(data not shown), which is most likely due to the possibility that 37G9and 19D6 are unable to block the interaction between B7x and itsreceptor(s).

Cancer-Expressed Human B7x Promotes Metastatic Cancer Progression InVivo

To facilitate the translation of this research into clinical trials,another lung metastatic cancer model was developed in which theexpression of human B7x on CT26 cancer cells significantly increasedmetastatic cancer progression in mice in vivo. A CT26 cell line,hB7x/CT26, was made that stably express human B7x on cell surface.Balb/c mice were iv injected with 1×10⁵/per-mouse of CT26 or hB7x/CT26and killed on day 17 to determine tumor nodules in the lung. FIG. 6shows that hB7x/CT26 resulted in much more tumor nodules in the lungthan CT26, 112.8 vs 23.8, demonstrating human B7x expressed on cancercells can promote metastatic cancer progression in vivo. This system canbe used to identify monoclonal antibodies that are suitable for humanclinical trials.

Monoclonal Antibody Inhibits Human B7x-Expressed Metastatic CancerProgression In Vivo

A lung metastatic cancer model was developed in which the expression ofhuman B7x on CT26 cancer cells (hB7x/CT26) significantly increasedmetastatic cancer progression in mice in vivo (FIG. 6). To furtherfacilitate the translation of this research into clinical trials, it wasdetermined whether mAbs were able to inhibit human B7x-expressedmetastatic cancer progression in vivo. Syngeneic BLAB/c mice were ivinjected with 1×10⁵/per-mouse of hB7x/CT26 on day 0 and then i.pinjected with 200 μg/per-mouse of mAb 1H3 on day 1,2,3,5,7,9,11,13,15,or PBS as the control. All mice were killed on day 17 and tumor nodulesin each lung were examined. Monoclonal antibody 1H3 had very goodtherapeutic effects, and reduced more than 60% of tumor nodules in thelung in such a robust humanized metastatic cancer model (FIG. 7).

Monoclonal Antibody-Dependent Cell-Mediated Cytotoxicity

mAbs were tested to determine whether they were able to act throughantibody-dependent cell-mediated cytotoxicity (ADCC). B7x/CT26 tumorcell as target cells, antibodies (0.2 μg/ml), and spleen cells fromsyngeneic BLAB/c mice as effector cells were incubated together at 37°C. for 4 hours, and then tumor cells were analyzed by flow cytometry.Compared to the control normal IgG, three (1H3, 15D2, 12D11) out of fourmAbs significantly killed B7x-expressed CT26 tumor cells (FIG. 8). Theseresults indicate that mAbs can kill B7x-positive tumor cells throughADCC.

B7x Protein is not Detected in Antigen Presenting Cells and T Cells

B7x protein is not detected in antigen-presenting cells (APC) and Tcells in both human and mice, as presented in Table 1.

TABLE 1 B7x protein is not detected in APCs and T cells before and afterstimuli. Immune cells Stimulation and disease models Human Dendriticcells CD40L, LPS, PMA+ ionomycin, cytokine cocktail Langerhans cellsCD40L, cytokine cocktail Monocytes LPS + IFN-γ B cells LPS, PMA+ionomycin T cells PHA, PMA+ ionomycin Mouse Dendritic cells LPS, IL-4,IFN-γ, IL-10, TNF-α, 4T1 cancer, S. pneumoniae infection, B. malayiinfection, Macrophages Thioglycolate, IL-4, LPS + IFN-γ, IL-6 + IL-10,TGF-β, Treg, 4T1 cancer, S. pneumoniae infection, B. malayi infection, Bcells LPS, anti-IgM F(ab′)2, PMA+ ionomycin, 4T1 cancer, S. pneumoniaeinfection T cells ConA, anti-CD3, PMA+ ionomycin, 4T1 cancer, S.pneumoniae infection Th1, Th2, Treg B7x protein was examined by flowcytometry with specific monoclonal or polyclonal Abs.

Mechanisms of Treatment for Cancers

The present results show that 1) cancer-expressed mice B7x promotesmetastatic cancer progression in two mice models and that monoclonalantibodies can inhibit metastatic cancer progression in vivo,demonstrating that blockage of cancer-associated B7x can be used as anovel treatment for metastatic cancers; and 2) human B7x expressed oncancer cells can also promote metastatic cancer progression in vivo andthat a monoclonal antibody can inhibit human B7x-expressing metastaticcancer progression in vivo, demonstrating that blockage ofcancer-associated human B7x can be used as a novel treatment formetastatic cancers. There are three possible mechanisms by which thistreatment can inhibit metastatic cancer progression in vivo (see FIG.9): (A) antibodies block the interaction between cancer-expressed B7xand activated T cells, therefore increasing T cell-mediated immunityagainst cancer; (B) antibodies bind cancer-expressed B7x and kill cancercells; and (C) antibodies block very low level of B7x expressed by sometissue cells, therefore inhibiting the expansion of regulatory T cells(Treg) and myeloid-derived suppressor cells (MDSC) within the tumormicroenvironment.

Discussion

The present technology has enormous market and clinical potential for atleast three reasons. 1) B7x is expressed in many human cancers, so it isan excellent therapeutic target for human cancers such as, for example,cancers of the lung, pancreas, kidney, brain, gut, prostate, breast,esophagus, skin, thyroid, stomach and ovary. 2) More than 90% of cancerpatients die from metastasis; therefore, therapies for metastasis aredesperately needed. 3) The antibodies describe herein recognize bothhuman and mice B7x, so these antibodies can be used in clinical trials.

Like B7x, CTLA-4 and PD-1 are two members of the B7/CD28 family. Anantibody against CTLA-4 (YERVOY® from Bristol-Myers Squibb Company) wasapproved by the FDA in March 2011 as a new drug for metastatic melanoma.Antibodies against PD-1 (MDX-1106) are in phase I clinical trials. Theseexisting technologies work by blockade of the B7/CD28 family membersCTLA-4 and PD-1 on activated T cells, so they increase immunity againstcancer but also induce autoimmune diseases. In contrast, the presenttechnology works by blockade of B7x on cancer cells, so as to increasespecific immunity against cancer without inducing autoimmune diseases.

Example II

Interaction of mAbs with B7x IgV Domain

Binding rate constants were estimated and corresponding equilibriumaffinity constants (K_(D)s) derived for the interactions of mAbs withrecombinant murine B7x ectodomain, as well as murine and human B7x-IgVthrough surface plasmon resonance (SPR). Antibodies 1H3, 12D11 and 15D12strongly interacted with all of these proteins, with dissociationconstants summarized in Table 2.

Anti-B7x mAb Therapy in Mouse B7x-Expressing Tumor Models

To develop a functional screening system for immunotherapy, tumor celllines expressing cell surface B7x were established, since most tumorlines were B7x protein negative. Mouse colon carcinoma CT26 (which wereB7x negative) and B7x/CT26 (which expressed mouse B7x on the surface)were intravenously (iv) injected into syngeneic BALB/c mice to induceexperimental lung metastasis. By day 17 after injection, the averagenumber of lung tumor nodules in the B7x/CT26 group was ˜3.5 fold higherthan that in the CT26 group, suggesting that the expression of B7x onCT26 tumor cells significantly promotes tumor progression in vivo.

The in vivo therapeutic effects of B7x-specific mAbs were screened inthe B7x/CT26-induced pulmonary metastasis model. B7x/CT26 cells were ivinjected into BALB/c mice followed by intraperitoneal (ip) injection ofanti-B7x mAbs. By day 17, lung tumor nodules were examined. Two mAbs,1H3 and 12D11, significantly reduced ˜60% of tumor nodules in lungs. The4T1 mammary carcinoma cell line that spontaneously metastasizes to thelung²⁰ was tried next. 1H3 treatment significantly reduced primarytumor-induced metastatic tumor nodules in the lung (FIG. 10B).

Anti-B7x mAb Therapy in a Human B7x-Expressing Tumor Model

Since both 1H3 and 12D11 recognize human B7x (Table 2), the therapeuticeffects of these two mAbs were tested in a human B7x-expressing tumormodel in vivo using hB7x/CT26, which expressed human B7x on mouse CT26.Like mouse B7x, the expression of human B7x on CT26 markedly increasedtumor nodules in the lung. Mice were iv injected with hB7x/CT26 and thentreated with 1H3 or 12D11. On day 17, the average numbers of lung tumornodules in 1H3-treated and 12D11-treated groups were 97 and 236,respectively, whereas the number in control group was 251 (FIG. 10C).Furthermore, 1H3 could recognize B7x expression on human colon and ovarycancers through immunohistochemistry. These results suggest that humanB7x promoted tumor growth in vivo and that mAb 1H3 recognized human B7xand inhibited human B7x expressing tumor progression in vivo. Since 1H3inhibited both human and mouse B7x-mediated tumor progression, it wasused for the subsequent experiments.

1H3 mAb-Treated Mice Generate Memory Response and Survive B7x/CT26Rechallenge

The effect of 1H3 on the survival of mice bearing B7x/CT26 tumors wasinvestigated. In agreement with the lung tumor nodule results, 1H3treated mice had a significantly lower mortality than controlIgG-treated mice. By day 60 post-injection of tumor, 100% of IgG-treatedmice were dead, whereas half of 1H3-treated mice remained alive (FIG.10D). Then, it was examined whether the surviving mice had generated amemory response to the tumor. These mice were rechallenged with the samenumber of B7x/CT26 cells, and all of them remained alive for thefollowing 60 days. On day 120, mice were sacrificed and hematoxylin andeosin (HE) staining of lung sections from these mice showed they werefree of tumor. These results suggest that the 1H3 treatment induced amemory response against the tumor.

Anti-B7x Therapy Increases Infiltrating T and NK Cells and DecreasesInfiltrating MDSCs within Tumors

To dissect the therapeutic mechanisms of 1H3 treatment, single-cellsuspensions were prepared from tumor-bearing lungs and immune cells wereanalyzed by flow cytometry. 1H3-treated mice had significantly higherpercentages of CD45+ immune cell infiltrate than control IgG-treatedmice (FIG. 11A). Among these CD45+ cells, the 1H3 treatment stronglyincreased infiltration of tumor by CD8 T cells and NK cells (FIG. 11B),two major types of anti-tumor immune cells. SPSYVYHQF/H(SEQ IDNO:5)-2L^(d) tetramer was used to detect CD8 T cells specific for CT26tumor antigen epitope AH1(amino acids 423-431 SPSYVYHQF (SEQ IDNO:5))^(21,22). In agreement with increased total CD8 T cells, 1H3treatment increased the percentage of AH1-specific CD8 T cells (FIG.11C). Recent studies identified the co-expression of Tim-3 and PD-1(Tim-3+PD-1+) cells as a unique phenotype of exhausted T cells inmelanoma and leukemia^(23,24). Therefore, the effect of 1H3 was examinedon these two inhibitory receptors on CD4 T cells. 1H3-treated mice hadsignificantly fewer CD4 T cells that were Tim-3+PD-1+, Tim-3+ alone andPD-1+ alone (FIG. 11D), suggesting 1H3 treatment reduce the conversionof CD4 T cells from activated to exhausted state. Along with thesefinding, the 1H3 treatment enhanced CD4 T cells to produce IFN-γ (FIG.11E), a critical cytokine for anti-tumor immunity.

In the tumor microenvironment, suppression of effector T cell functionis often driven by immunosuppressive cells. Therefore, the effect of 1H3treatment on immunosuppressive cell infiltrates was investigated intumor-bearing lungs. The treatment did not change the percentage ofFoxp3+CD4+ regulatory T cells (Tregs), but reduced CD11b+Ly6C+ monocyticmyeloid-derived suppressor cells (MDSCs) infiltrating tumor (FIG. 11F).The combined increase of CD8 T cell, NK and IFN-γ-producing CD4 T cellsand reduction of MDSCs in the 1H3 treatment afforded a significantlylower ratio of the suppressive MDSCs and Tregs to effector anti-tumorimmune cells (FIG. 11G).

Anti-B7x Therapy Decreases VEGF and TGF-β in the Tumor Microenvironment

VEGF stimulates angiogenesis in the tumor microenvironment andfacilitates tumor growth and metastasis²⁵⁻²⁸. VEGF concentration intumor-bearing lungs from 1H3 treated mice was significantly lower thanthat of control mice (FIG. 12A). Correspondingly, CD31 expressionpattern in tumor vasculature revealed that the 1H3 treatment inhibitedintratumor vasculature (FIG. 12B). The 1H3 treatment also lowered theconcentration of TGF-β in tumor-bearing lungs (FIG. 12C), one of the keycytokines responsible for suppressing anti-tumor responses^(29,30).

1H3 Kills Tumor Cells Through ADCC but not CDC

One way in which antibodies can eliminate virus-infected cells or tumorcells is via antibody-dependent cellular cytotoxicity (ADCC)³¹⁻³³. Itwas examined whether 1H3 kills tumor cells through ADCC. 1H3 induceddeath of 50% more target cells compared to control IgG (FIG. 13A). Inagreement with the in vitro ADCC result, significantly increased numbersof apoptotic cells were present in tumors from 1H3 treated mice (FIG.13B). Antibodies can also eliminate tumor cells via complement-dependentcytotoxicity (CDC)³⁴; however, 1H3 specific CDC activity could not bedetected (data not shown).

1H3 Blocks B7x-Mediated T Cell Coinhibition

T cells proliferated vigorously when incubated with anti-CD3 and controlIg with more than 73% of T cells dividing. When T cells were incubatedwith anti-CD3 and B7x-Ig, significantly fewer T cells proliferated, withabout 41% dividing. The presence of 1H3 in the system significantlyneutralized B7x-mediated T cell coinhibition, as 1H3 increased T cellproliferation to >61%. Furthermore, Fab fragment of 1H3 had a similarneutralizing effect on B7x-induced T cell coinhibition. These resultsreveal that 1H3 can partially block B7x-mediated T cell coinhibition. Toassess whether 1H3 therapy depends on ADCC and/or functionalneutralization in vivo, therapeutic efficacies between 1H3 and its Fab(which cannot cause ADCC) were compared. Mice treated with the Fab hadsignificantly fewer lung tumor nodules than did mice treated withcontrol IgG, but had significantly more lung tumor nodules than did micetreated with 1H3 (FIG. 13C). Taken together, these results suggest that1H3 inhibits tumor growth through the combination of ADCC and functionalneutralization.

TABLE 2 Surface plasmon resonance measurements. mAb k_(on) (M⁻¹ · s⁻¹)k_(off) (s⁻¹) K_(D) (nM) murine B7x (IgV domain) 1H3 2.455(4)^(a) × 10⁶ 0.001248(7) 0.508(3)  12D11 2.140(4) × 10⁶  0.001341(8) 0.627(4)  15D121.790(3) × 10⁶  0.001135(7) 0.634(4)  murine B7x 1H3 6.71(3) × 10⁵0.001298(7) 1.94(1) 12D11 6.62(4) × 10⁵ 0.001177(7) 1.78(1) 15D124.40(3) × 10⁵ 0.001095(6) 2.49(2) human B7x (IgV domain) 1H3 2.53(2) ×10⁵  0.00917(4) 36.2(3) 12D11 2.13(1) × 10⁵  0.00928(3) 43.5(3) 15D121.388(7) × 10⁵   0.01039(2) 74.9(4) ^(a)The value in parentheses denotesthe standard error in the last digit

Example III

Anti-B7x Antibody Therapy is Better than Anti-PD-1 Antibody Therapy inTwo Lung Metastasis of Cancer Models

Anti-B7x antibody therapy was compared to anti-programmed cell deathprotein 1 (PD-1) antibody therapy in two lung metastasis of cancermodels. Anti-PD-1 therapy is currently in phase 1 clinical trials incancer patients.

In one model, BALB/c mice were iv injected with B7x/CT26 tumor on day 0and then ip injected with normal IgG (control), anti-B7x mAb 1H3, oranti-PD-1 mAb RMP1-14 on day 1,3,7,11, 14. On day 17, mice weresacrificed and numbers of lung tumor nodules were evaluated. Anti-B7xtreatment reduced more than 58% of lung tumor nodules, ***P<0.001;whereas anti-PD-1 treatment reduced only 34% of lung tumor nodules anddid not reach statistical significance (FIG. 14A).

In a second model, BALB/c females mice were injected with B7x/4T1 tumorinto the mammary fatpad on day 0 and then ip injected with normal IgG(control), anti-B7x mAb 1H3, or anti-PD-1 mAb RMP1-14 on day 8,11,13,15,18. On day 20, mice were sacrificed and numbers of lung tumor noduleswere evaluated. Anti-B7x treatment reduced more than 58% of lung tumornodules, *P<0.05; whereas anti-PD-1 did not have an effect (FIG. 14B).

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1.-26. (canceled)
 27. A method for treating metastatic cancer in apatient having metastatic cancer or for preventing metastasis in acancer patient at risk for metastasis comprising administering to thepatient a IgG1 monoclonal antibody, or a fragment thereof, that binds toB7x, in an amount effective to treat or prevent metastasis in a patient,wherein the IgG1 monoclonal antibody or fragment binds to amino acidresidues 35-148 of SEQ ID NO:1, wherein the IgG1 monoclonal antibody orfragment kills tumor cells through antibody-dependent cellularcytotoxicity.
 28. The method of claim 27, wherein the IgG1 monoclonalantibody or fragment blocks inhibition of T cell function by binding toB7x.
 29. The method of claim 27 comprising determining a first level ofB7x expression in a tumor sample from the patient, and comparing thefirst level of B7x expression in the tumor sample to a second level ofB7x expression in a corresponding healthy tissue.
 30. The method ofclaim 27, wherein the patient has metastatic cancer.
 31. The method ofclaim 27, wherein the patient is a cancer patient at risk formetastasis.
 32. The method of claim 27, wherein the cancer is a cancerof the skin, breast, pancreas, prostate, ovary, kidney, esophagus,gastrointestinal tract, colon, brain, liver, lung, head and/or neck. 33.The method of claim 27, wherein administration of the IgG1 monoclonalantibody or fragment thereof decreases the number of tumor nodules inthe patient.
 34. The method of claim 27, wherein administration of theIgG1 monoclonal antibody or fragment thereof reduces the number ofmetastases.
 35. The method of claim 27, wherein administration of theIgG1 monoclonal antibody or fragment thereof prevents the occurrence orreoccurrence of metastasis.
 36. The method of claim 27, whereinadministration of the IgG1 monoclonal antibody or fragment thereofincreases the patient's survival time.
 37. The method of claim 27,wherein the IgG1 monoclonal antibody or fragment thereof does notinclude an antibody-partner molecule conjugate.
 38. The method of claim27, wherein the IgG1 monoclonal antibody or fragment thereof is the soletherapeutic anti-cancer agent administered to the patient.
 39. Themethod of claim 27, wherein the IgG1 monoclonal antibody or fragmentthereof is administered in combination with another anti-cancer agent.40. The method of claim 27, wherein administration of the IgG1monoclonal antibody or fragment thereof prevents the reoccurrence of atumor in the patient.
 41. The method of claim 27, further comprisingadministering to the patient a second anti-cancer agent.
 42. The methodof claim 41, wherein the second anti-cancer agent is selected from thegroup consisting of an antibody against CTLA-4, an antibody againstPD-1, an anti-EGFR agent, an alkylating agent, paclitaxel, docetaxel,and a topoisomerase inhibitor.
 43. The method of claim 42, wherein thesecond anti-cancer agent is an antibody against CTLA-4.
 44. The methodof claim 42, wherein the anti-EGFR agent is selected from the groupconsisting of panitumumab, cetuximab, gefitinib and erlotinib.
 45. Themethod of claim 42, wherein the alkylating agent is selected from thegroup consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin,satraplatin, triplatin tetranitrate, mechlorethamine, cyclophosphamide,chlorambucil and ifosfamide.
 46. The method of claim 42, wherein thetopoisomerase inhibitor is selected from the group consisting ofirinotecan, topotecan, amsacrine, etoposide, etoposide phosphate andteniposide.
 47. A method for preventing reoccurrence of a tumor in apatient comprising administering to the patient a IgG1 monoclonalantibody, or a fragment thereof, that binds B7x, in an amount effectiveto prevent reoccurrence of a tumor in a patient, wherein the IgG1monoclonal antibody or fragment thereof binds to amino acid residues35-148 of SEQ ID NO:1, wherein the IgG1 monoclonal antibody or fragmentthereof kills tumor cells through antibody-dependent cellularcytotoxicity, and wherein the antibody or antibody fragment blocksinhibition of T cell function by B7x.